Antibody identification by lineage analysis

ABSTRACT

A method of screening is provided. In certain embodiments, the method involves a) obtaining the nucleotide sequences of: i. a heavy chain-encoding nucleic acid that encodes the variable domain of a heavy chain of a first antibody of an animal; and ii. a light chain-encoding nucleic acid that encodes the variable domain of a light chain of the first antibody; b) obtaining nucleotide sequences of cDNAs encoding at least a portion of the antibody repertoire of the animal; c) computationally screening the sequences obtained in b) to identify heavy and light chain sequences that are related by lineage to the heavy and light chain sequences of a); and d) testing at least one pair of the heavy and light chain sequences identified in c) to identify a second antibody that binds to the same antigen as the first antibody.

FIELD OF THE INVENTION

The invention relates to monoclonal antibodies.

BACKGROUND TO THE INVENTION

Antibodies are proteins that bind a specific antigen. Generally,antibodies are specific for their targets, have the ability to mediateimmune effector mechanisms, and have a long half-life in serum. Suchproperties make antibodies powerful therapeutics. Monoclonal antibodiesare used therapeutically for the treatment of a variety of conditionsincluding cancer, inflammation, and other diseases. There are currentlyover two dozen therapeutic antibody products on the market and hundredsin development. There is a constant need for new antibodies havingdesirable properties and methods for isolating the same.

It is understood that an the full antibody repertoire of an immunizedsubject (also referred as “immunized antibody repertoire” or “immunizedrepertoire”) contains a very large number of antibodies having sequencediversity which can, in some cases, impart variations with respect toaffinity, specificity and in vivo functionality. In view of this it issometimes desirable to sample a significant number of antibodies from asubject to identify antibodies that possess similar or improvedproperties relative to, e.g., an antibody of interest. Therefore thereis a need in the art for the identification of new antibodies in adirect and efficient manner for use in the development of therapeuticcandidates, in diagnostic applications and/or research products.

US20110065112 describes a method for identifying lineage-relatedantibodies. In some embodiments this method involves: obtaining theantibody sequences from a population of B cells; grouping the antibodysequences to provide a plurality of groups of lineage-relatedantibodies; testing a single antibody from each of the groups in abioassay and, after the first antibody has been identified, testingfurther antibodies that are in the same group as the first antibody in asecond bioassay. US20110065112 also describes a method that involvestesting a plurality of antibodies obtained from a first portion of anantibody producing organ of an animal; obtaining the sequence of a firstidentified antibody; obtaining from a second portion of said antibodyproducing organ the sequences of further antibodies that are related bylineage to said first antibody; and, c) testing the further antibodiesin a second bioassay.

US20100204059 describes a method for obtaining nucleic acid encoding aplurality of antibodies. In certain embodiments, the method comprisesobtaining from an immunized animal nucleic acid encoding the amino acidsequence of the heavy and light chains of a second antibody that bindsto the antigen as a first antibody and differs in amino acid sequence tothe first antibody, wherein the obtaining is done by amplificationusing: i. a first primer pair that includes oligonucleotides arecomplementary to CDR-encoding regions first antibody.

The inventors have developed a new method that serves to identifylineage-related antibodies from an antibody repertoire of an immunizedanimal, based on the properties of an antibody of interest usingcomputational means.

SUMMARY OF THE INVENTION

According to a first aspect of the invention a method of screening isprovided. In certain embodiments, the method involves

a) obtaining the nucleotide sequences of: i. a heavy chain-encodingnucleic acid that encodes the variable domain of a heavy chain of afirst antibody of an animal; and ii. a light chain-encoding nucleic acidthat encodes the variable domain of a light chain of the first antibody;b) obtaining nucleotide sequences of cDNAs encoding at least a portionof the antibody repertoire of the animal;c) computationally screening the sequences obtained in b) to identifyheavy and light chain sequences that are related by lineage to the heavyand light chain sequences of a); and d) testing at least one pair of theheavy and light chain sequences identified in c) to identify a secondantibody that binds to the same antigen as the first antibody.

According to a further aspect of the invention there is provided amethod of producing an antibody, the method comprising

a) obtaining the nucleotide sequences of: i. a heavy chain-encodingnucleic acid that encodes the variable domain of a heavy chain of afirst antibody of an animal; and ii. a light chain-encoding nucleic acidthat encodes the variable domain of a light chain of the first antibody;b) obtaining nucleotide sequences of cDNAs encoding at least a portionof the antibody repertoire of the animal;c) computationally screening the sequences obtained in b) to identifyheavy and light chain sequences that are related by lineage to the heavyand light chain sequences of a);d) introducing at least one pair of the heavy and light chain sequencesobtained in c) into a host cell;e) incubating the host cell to permit expression of the antibody;f) purifying the antibody expressed in e) to produce a second antibodythat binds to the same antigen as the first antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram illustrating one embodiment of the invention.

DEFINITIONS

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” includes a plurality of such antibodies and reference to “aframework region” includes reference to one or more framework regionsand equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regions(VL and VH respectively) are together responsible for binding to anantigen, and the constant regions are responsible for the antibodyeffector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa, or about 200 to 225amino acids, or about 214 amino acids) comprise a variable region ofabout 110 amino acids at the NH₂-terminus and a kappa or lambda constantregion at the COOH-terminus. Full-length immunoglobulin “heavy chains”(of about 50 kDa, or about 440 to 460 amino acids), or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986)).

An immunoglobulin light or heavy chain variable region consists of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see,“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services, (1991)). The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs. The CDRs are primarilyresponsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, from antibodyvariable and constant region genes belonging to different species. Forexample, the variable segments of the genes from a mouse monoclonalantibody may be joined to human constant segments, such as gamma 1 andgamma 3. An example of a therapeutic chimeric antibody is a hybridprotein composed of the variable or antigen-binding domain from a rabbitantibody and the constant or effector domain from a human antibody(e.g., the anti-Tac chimeric antibody made by the cells of A.T.C.C.deposit Accession No. CRL 9688), although other mammalian species may beused.

As used herein, the term “humanized antibody” or “humanizedimmunoglobulin” refers to a non-human (e.g., mouse or rabbit) antibodycontaining one or more amino acids (in a framework region, a constantregion or a CDR, for example) that have been substituted with acorrespondingly positioned amino acid from a human antibody. In general,humanized antibodies produce a reduced immune response (ornon-immunogenic) in a human host, as compared to a non-humanized versionof the same antibody.

It is understood that the humanized antibodies designed and produced bythe present method may have additional conservative amino acidsubstitutions which have substantially no effect on antigen binding orother antibody functions. By conservative substitutions is intendedcombinations such as those from the following groups: gly, ala; val,ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Aminoacids that are not present in the same group are “substantiallydifferent” amino acids.

The term “specific binding” refers to the ability of an antibody topreferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

In certain embodiments, the affinity between a capture agent (antibody)and analyte when they are specifically bound in a capture agent/analytecomplex is characterized by a K_(D) (dissociation constant) of less than10⁻⁶M, less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than10⁻⁹ M, less than 10⁻¹¹ M, or less than about 10⁻¹² M.

A “variable region” of a heavy or light antibody chain is an N-terminalmature domain of the chains. All domains, CDRs and residue numbers areassigned on the basis of sequence alignments and structural knowledge.Identification and numbering of framework and CDR residues is asdescribed in by Kabat, Chothia (Chothia Structural determinants in thesequences of immunoglobulin variable domain. J Mol Biol 1998;278:457-79) or others.

VH is the variable domain of an antibody heavy chain. VL is the variabledomain of an antibody light chain, which could be of the kappa (K) or ofthe lambda isotype. K-1 antibodies have the kappa-1 isotype whereas K-2antibodies have the kappa-2 isotype.

A “buried residue” is an amino acid residue whose side chain has lessthan 50% relative solvent accessibility, which is calculated as thepercentage of the solvent accessibility relative to that of the sameresidue, X, placed in an extended GGXGG peptide. Methods for calculatingsolvent accessibility are well known in the art (Connolly 1983 J. appl.Crystallogr, 16, 548-558).

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;fusion proteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like. Polypeptides may be of any size, and theterm “peptide” refers to polypeptides that are 8-50 residues (e.g., 8-20residues) in length.

As used herein the term “isolated,” when used in the context of anisolated antibody, refers to an antibody of interest that is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, and even at least 99% free from other components withwhich the antibody is associated with prior to purification.

The terms “treatment”, “treating” and the like are used herein to referto any treatment of any disease or condition in a mammal, e.g.particularly a human or a mouse, and includes: a) preventing a disease,condition, or symptom of a disease or condition from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; b) inhibiting a disease, condition, or symptomof a disease or condition, e.g., arresting its development and/ordelaying its onset or manifestation in the patient; and/or c) relievinga disease, condition, or symptom of a disease or condition, e.g.,causing regression of the condition or disease and/or its symptoms.

The terms “subject,” “host,” “patient,” and “individual” are usedinterchangeably herein to refer to any mammalian subject for whomdiagnosis or therapy is desired, particularly humans. Other subjects mayinclude cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses,and so on.

“Corresponding amino acids”, as will be described in greater detailbelow, are amino acid residues that are at an identical position (i.e.,they lie across from each other) when two or more amino acid sequencesare aligned. Methods for aligning and numbering antibody sequences areset forth in great detail in Chothia, supra, Kabat supra, and others. Asis known in the art (see, e.g. Kabat 1991 Sequences of Proteins ofImmunological Interest, DHHS, Washington D.C.), sometimes one, two orthree gaps and/or insertions of up to one, two, three or four residues,or up to about 15 residues (particularly in the L3 and H3 CDRs) may bemade to one or both of the amino acids of an antibody in order toaccomplish an alignment.

A “natural” antibody is an antibody in which the heavy and lightimmunoglobulins of the antibody have been naturally selected and pairedby the immune system of a multi-cellular organism, as opposed tounnaturally paired antibodies made by e.g. phage display. As such, thesubject parental antibodies do not usually contain any viral (e.g.,bacteriophage M13)-derived sequences. Spleen, lymph nodes and bonemarrow are examples of tissues that produce natural antibodies. Forexample, the antibodies produced by the antibody producing cellsisolated from an animal immunized with an antigen are naturalantibodies.

The term “non-naturally paired”, with respect to VH and VL chains of anengineered antibody, refers to an antibody that contains a naturallyoccurring VH sequence and a naturally occurring VL sequence, where thesequences are not paired with one another naturally, i.e., in a naturalantibody. Thus, a non-naturally paired antibody is a combination of VHand VL chain of two different natural antibodies. The VH and VL chainsof a non-naturally paired antibody are not mutated relative to the VHand VL chains of the two different antibodies which provided the VH andVL chains. For example, the “non-naturally paired” IgH and IgL chains ofthe engineered antibody may contain the IgH variable chain from a firstantibody producing cell obtained from an animal and the IgL variablechain of second antibody producing cell obtained from the same animal,where the amino acid sequence of the antibody produced by the first cellis different from the amino acid sequence of the antibody produced bythe second cell. In this example, the IgH and IgL chains may be from thesame lineage group. An antibody containing “non-naturally paired” IgHand IgL chains may or not be made by phage display. As such, antibodiesmay or may not contain viral (e.g., bacteriophage M13)-derivedsequences.

The term “lineage-related antibodies” and “antibodies that are relatedby lineage” as well as grammatically-equivalent variants thereof, areantibodies that are produced by cells that share a common B cellancestor or convergence toward similar sequence during affinitymaturation. Related antibodies produced by related antibody producingcells bind to the same epitope of an antigen and are typically verysimilar in sequence, particularly in their L3 (i.e., the light chainCDR3) and H3 (heavy chain CDR3) regions. Both the H3 and L3 CDRs ofantibodies that are related to one another by lineage have an identicallength and a near identical sequence (i.e., differ by up to 5, i.e., 0,1, 2, 3, 4 or 5 residues). The VH or VL domains of antibodies within arelated group of antibodies may have amino acid sequences that are atleast about 80% identical (e.g., at least 85% identical, at least 90%identical, at least 95% or at least 98% or at least 99% identical),ignoring any gaps or insertions made to facilitate alignment of thesequences. In certain cases, the B cell ancestor contains a genomehaving a rearranged light chain VJC region and a rearranged heavy chainVDJC region, and produces an antibody that has not yet undergoneaffinity maturation. “Naïve” or “virgin” B cells present in spleentissue, are exemplary B cell common ancestors. Related antibodies arerelated via a common antibody ancestor, e.g., the antibody produced inthe naïve B cell ancestor. The term “related antibodies” is intended todescribe a group of antibodies that are produced by cells that arisefrom the same ancestor B-cell. A “lineage group” contains a group ofantibodies that are related to one another by lineage.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the subject method is illustrated in FIG. 1. Withreference to FIG. 1, the method may involve obtaining the cDNA sequencesfor a first antibody from an animal. As would be apparent, this stepinvolves obtaining the nucleotide sequences of: i. a VH cDNA, whereinthe VH cDNA encodes the variable domain of a heavy chain of the firstantibody; and ii. a VL cDNA, wherein the VL cDNA encodes the variabledomain of a light chain of the first antibody. In addition, the methodinvolves obtaining the nucleotide sequences of cDNAs, i.e., heavy andlight chain cDNAs, encoding at least a portion of the antibodyrepertoire of the same animal (i.e., the same individual) to producereference sequences. These cDNAs may be obtained in bulk preparationfrom mRNA prepared from a population of antibody-producing cells, e.g.,spleen or plasma cells, isolated from the animal. In many cases, thesequences obtained in this step may be deposited into a database, andoptionally annotated as to whether they are heavy chain or light chainsequences. In the next step, the reference sequences, i.e., thesequences obtained by sequencing the repertoire of the animal, arescreened computationally to identify heavy and light chain sequencesthat are related by lineage to the heavy and light chain sequences ofthe first antibody. Such antibodies generally have very similarsequences, and have H3 CDRs of identical length and near identicalsequence as well as L3 CDRs of identical length and a near identicalsequence. This step may be done by comparing a window (e.g., at least20, 30, 50, 80 or 100 contiguous amino acids) of the heavy and lightchain of the first antibody with the reference sequences to identifysimilar sequences. Alternatively, this step may be done by scanning thereference sequences to identify those that encode sequences that aresimilar or identical to the heavy chain CDR3 and light chain CDR3sequences of the first antibody. In this embodiment, the initial stepsof this part of the method may be done by comparing only the heavy andlight chain CDR3 sequences of the first antibody to the referencesequences. This step may result in a list of heavy chain sequences and alist of light chain sequences, where the heavy chain sequences on thelist are from heavy chains of antibodies that are related by lineage tothe heavy chain of the first antibody and the light chain sequences onthe list are from light chains of antibodies that are related by lineageto the light chain of the first antibody. Depending on how the librarypreparation and sequencing is done, the heavy and light chain sequencesmay already be paired in the sense that when a heavy chain sequence isidentified, the light chain to which that heavy chain is naturallypaired may be apparent. In other embodiments, there may be noinformation about how the heavy and light chains are naturally paired.Once the list of heavy chain sequences and the list of light chainsequences have been created, the method involves testing at least onepair of the heavy and light chain sequences to identify a secondantibody that binds to the same antigen as the first antibody. In somecases, this may involve amplifying cDNA sequences encoding the heavy andlight chains by PCR from a pool of cDNA (e.g., the same pool of cDNAthat was sequenced earlier in the method) and expressing the amplifiedcDNAs in a cell. In other embodiments, the heavy and light chainsequences can be made synthetically and expressed in a cell. In someembodiments, the testing step may comprises testing at least 10% of allpossible combinations of the identified heavy and light chains forbinding to the antigen. In particular embodiments, the heavy and lightchain sequences tested may be randomly selected from the identifiedheavy and light chain sequences.

The present method provides a way to efficiently screen millions or tensof millions of antibody sequences without having to screen each of theantibodies individually as is done in conventional methods. The methodallows one to home in on a group of heavy chain sequences and a group oflight chain sequences, the individual sequences of which can be testedin a pairwise manner and, optionally, mutated (e.g., using the methodsset out in US20050033031) with the knowledge that most or all of thecandidate antibodies are capable of binding to the target antigen. Thecandidate antibodies are expected to vary in their biochemical and/orpharmacological properties (e.g., they may vary in their affinity,specificity and/or in vivo functionality, etc.). As such, after theyhave been produced, the candidate antibodies can be tested for adesirable property.

Some embodiments of the present method allow one to harness and make useof the diversity of sequences in an antibody repertoire in a way that isinaccessible by other methods.

For example, methods in which phylogenetically-related antibodies areidentified by PCR using primers to the CDR regions can be limitedbecause, in some cases, the primers actually overwrite the sequencesthat provide antibody diversity. For example, if a CDR3 primer is used,any sequence mismatches in the CDR3 region will be overwritten by theprimer, resulting in a PCR product that does not actually reflect thediversity of the original antibody population. The resultant PCR productmay encode a functional antibody but the sequences variations at theprimer binding site may have been overwritten. In other words, theprimers used in several PCR-based methods are specifically designed tobind to sequence that are modified during affinity maturation. Use ofsuch primers will frequently not result in a PCR product that reflectsthe original sequence.

Further, in some cases, PCR-based methods can only, at best, amplify afragment encoding only part of a heavy or light chain (e.g., sequencesthat are 5′ or 3′ to one of the primers) rather than, for example, theentire variable domain of a heavy or light chain. While one canpotentially piece together the two ends of a heavy or light chain codingsequence to produce a functional sequence (e.g., by performing anotherround of PCR to obtain an overlapping fragment and then joining thefragments together) this additional step adds significant technicalcomplexity to the method and, because the initial PCR reactions are doneon a pool of sequences, the 5′ end of the heavy or light chain from oneantibody can become be fused with a 3′ end sequence from anotherantibody.

Finally, the sequence comparison algorithms used in the present methodcan be tuned to identify phylogenetically distant family members (e.g.,family members that have, e.g., CDR3 regions with more than 3, 4, or 5amino acid substitutions relative to the test sequence) that may not beamplified using PCR-based strategies. In these methods, the sequencecomparison step could use an algorithm that identifies approximatepatterns rather than exact patterns (e.g., a fuzzy match algorithm orthe like) to identify sequences that have CDR sequences that aresignificantly different from the reference sequence. In contrast,CDR-based PCR may narrow the analysis down for example by introducing abias meaning more distant family members will be missed as aconsequence. This method may be particularly informative should geneconversion shift antigen reactivity away from the reference sequence toanother lineage.

Many warm-blooded animals, in particular mammals such as humans,rabbits, mice, rats, sheep, cows, pigs, goats, horse, camel and ayessuch as chickens and turkeys, may be used as a source ofantibody-producing cells. However, in certain embodiments a rabbit ormouse is used because of their ease in handling, well-defined genetictraits, and the fact that they may be readily sacrificed. Procedures forimmunizing animals are well known in the art, and are described inHarlow et al., (Antibodies: A Laboratory Manual, First Edition (1988)Cold Spring Harbor, N.Y.). In some embodiments the antibody repertoireof the animal may be obtained by sequencing cDNAs encoding heavy andlight chains made from splenocytes of the animal. In other embodiments,the method may involve sequencing cDNAs encoding heavy and light chainsmade from circulating B cells of the animal.

In some embodiments, the animal may be been immunized with the antigen,e.g., multiple times in the presence of an adjuvant. In theseembodiments, suitable antigens include extracellularly-exposed fragmentsof Her2, GD2, EGF-R, CEA, CD52, CD20, Lym-1, CD6, complement activatingreceptor (CAR), EGP40, VEGF, tumor-associated glycoprotein TAG-72 AFP(alpha-fetoprotein), BLyS (TNF and APOL-related ligand), CA125(carcinoma antigen 125), CEA (carcinoembrionic antigen), CD2 (T-cellsurface antigen), CD3 (heteromultimer associated with the TCR), CD4,CD11a (integrin alpha-L), CD14 (monocyte differentiation antigen), CD20,CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD25 (IL-2receptor alpha chain), CD30 (cytokine receptor), CD33 (myeloid cellsurface antigen), CD40 (tumor necrosis factor receptor), CD44v6(mediates adhesion of leukocytes), CD52 (CAMPATH-1), CD80 (costimulatorfor CD28 and CTLA-4), complement component C5, CTLA, EGFR, eotaxin(cytokine A11), HER2/neu, HLA-DR, HLA-DR10, HLA ClassII, IgE, GPiib/iiia(integrin), Integrin aVβ3, Integrins a4β1 and a4β7, Integrin β2,IFN-gamma, IL-1β, IL-4, IL-5, IL-6R (IL6 receptor), IL-12, IL-15, KDR(VEGFR-2), lewisy, mesothelin, MUC1, MUC18, NCAM (neural cell adhesionmolecule), oncofetal fibronectin, PDGFβR (Beta platelet-derived growthfactor receptor), PMSA, renal carcinoma antigen G250, RSV, E-Selectin,TGFbeta1, TGFbeta2, TNFalpha, TRAIL-R1, VAP-1 (vascular adhesion protein1), TNFα, or the like. In many embodiments, a peptide having the aminoacid sequence corresponding to a portion of an extracellular domain ofone of the above-listed proteins may be employed as an antigen.

In other embodiments, the animal may have an autoimmune disease, or hasdeveloped resistance to or has recovered from a disease (e.g., cancer).In some embodiments, antibody-producing cells may also be obtained froma subject that has generated the cells during the course of a selecteddisease or condition. For instance, antibody-producing cells from ahuman with a disease of unknown cause, such as rheumatoid arthritis, maybe obtained and used in an effort to identify antibodies which have aneffect on the disease process or which may lead to identification of anetiological agent or body component that is involved in the cause of thedisease. Similarly, antibody-producing cells may be obtained fromsubjects with disease due to known etiological agents such as malaria orAIDS. These antibody-producing cells may be derived from the blood,lymph nodes or bone marrow, as well as from other diseased or normaltissues. Antibody producing cells may also be prepared from bloodcollected with an anticoagulant such as heparin or EDTA. Theantibody-producing cells may be further separated from erythrocytes andpolymorphs using standard procedures such as centrifugation withFicoll-Hypaque (Pharmacia, Uppsula, Sweden). Antibody-producing cellsmay also be prepared from solid tissues such as lymph nodes or tumors bydissociation with enzymes such as collagenase and trypsin in thepresence of EDTA.

The cDNAs encoding the first antibody may be obtained by any suitablemeans. In particular embodiments, the first antibody may be obtained byconventional hybridoma technology. In other embodiments, however, themethod may employ flow cytometry (FACS) of cell populations obtainedfrom rabbit spleen, bone marrow, lymph node, plasma or other lymphorgans, e.g., through incubating the cells with labeled antigen andsorting the labeled cells using a cell sorter. The first antibody mayalso be identified by B cell cloning, i.e., screening individual B cellsfor an antibody of interest, and then amplifying the coding sequences ofthat antibody from the B cell from which the antibody is secreted,before the B cells dies and/or the mRNA encoding the antibody becomesdegraded. A number of methods can be used to identify an antibody ofinterest, and clone its cDNA. In an exemplary embodiment, nucleic acidsencoding the VH and VL domains of an antibody are isolated from anantibody-producing cell, e.g., a hybridoma. In order to produceantibody-producing hybridoma, an animal can be immunized with an antigenand once a specific immune response of the rabbit has been established,cells from the spleen of the immunized animal are fused with a suitableimmortal cell (e.g., NIH 3T3, DT-40 or 240E cell, etc.; Spieker-Polet etal, Proc. Natl. Acad. Sci. 92: 9348-9352, 1995) to produce hybridomacells. Supernatants from these hybridoma cells are screened for antibodysecretion by enzyme-linked immunosorbent assay (ELISA) and positiveclones secreting monoclonal antibodies specific for the antigen can beselected and expanded according to standard procedures (Harlow et al.,Antibodies: A Laboratory Manual, First Edition (1988) Cold springHarbor, N.Y.; and Spieker-Polet et al., supra).

In some embodiments, the antibody-producing cells may be obtained fromthe animal at a time when the transit of plasma cells in the blood ismaximal. In some cases, this may be done shortly after (i.e., within 2to 14 days of, e.g., 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12,11-13 or 12-14 days of) the initial immunization, the secondaryimmunization or a tertiary immunization. In any embodiment, the cellsmay be plated in a minimal amount of culture medium at a single celldilution, maintained in culture for a period of time (e.g., 1-7 days)and culture medium may be tested for antibodies that react with theantigen before the plasma cells die. In any embodiment, the plated cellsmay be stimulated to produce more antibody while they are in culture.These embodiments do not require that the plasma cells divide in culturehowever, in some embodiments, the plasma cells may divide for a fewgenerations. In these embodiments, the B cells can be separated fromother cells by FACS prior to plating at single cell dilution.

As would be recognized, the cDNAs from the single cells may be barcodedso that they can be mixed together, optionally with other templates, andsequenced. In an exemplary embodiment, the heavy and light chain cDNAsare both barcoded with the same sequence, thereby allowing the sequencesfor those cDNAs to be paired with one another after the cDNAs have beenmixed with other templates and sequenced.

In particular embodiments, the animal may be immunized by multipleantigens (e.g., at least 2, at least 5, at least 10, at least 50, atleast 100, at least 500 or at least 1,000, up to 5,000 or moreantigens).

Suitable monoclonal antibodies may be further selected on the basis ofbinding activity, including its binding specificity, binding affinity,binding avidity, a blocking activity or any other activity that causesan effect (e.g. promoting or inhibiting a cellular phenotype, e.g., cellgrowth, cell proliferation, cell migration, cell viability (e.g.,apoptotis), cell differentiation, cell adherence, cell shape changes(e.g., tubular cell formation), complement dependent cytotoxicity CDC,antibody-dependent cell-mediated cytotoxicity ADCC, receptor activation,gene expression changes, changes in post-translational modification(e.g., phosphorylation), changes in protein targeting (e.g., NFκBlocalization etc.), etc., or inhibition of receptor multimerization(e.g., dimer or trimerization) or receptor-ligand interactions). Inother embodiments, an affinity purification method maybe utilized toisolate antibody producing cells that produce antibodies that bind to anantigen. The antigen with which the animal was immunized may beimmobilized on a solid phase and used to selectively retain antibodyproducing cells that express an antibody on their surface that binds tothe antigen, while other cells are washed away. The retained cells maythen be eluted by a variety of methods, such as by using an excess ofthe antigen, chaotropic agents, changing the pH, salt concentration,etc. Any of the well-known methods for immobilizing or coupling antigento a solid phase may be used. For example, when the antigen is a cancercell, appropriately treated microtiter plate that will bind to cells maybe used, such as microtiter plates for cell culture. In the instanceswhere the antigen is a protein, the protein may be covalently attachedto a solid phase, for example, sepharose beads, by well-knowntechniques, etc. Alternatively, a labeled antigen may be used tospecifically label cells that express an antibody that binds to theantigen and the labelled cells may then be isolated by cell sorting(e.g., by FACS). In certain cases, methods for antibody purification maybe adapted to isolate antibody producing cells. Such methods are knownand are described in, for example, J Immunol Methods. 2003 November;282(1-2):45-52; J Chromatogr A. 2007 Aug. 10; 1160(1-2):44-55; J BiochemBiophys Methods. 2002 May 31; 51 (3):217-31. Cells may also be isolatedusing magnetic beads or by any other affinity solid phase capturemethod, protocols for which are known. In some embodiments,antigen-specific antibody producing cells may be obtained from blood byflow cytometry using the methods described in Wrammert (Nature 2008 453:667-672), Scheid (Nature 2009 458: 636-640), Tiller (J. Immunol. Methods2008 329 112-124) or Scheid (Proc. Natl. Acad. Sci. 2008 105:9727-9732), for example, which are incorporated by reference fordisclosure of those methods. Exemplary antibody-producing cellenrichment methods include performing flow cytometry (FACS) of cellpopulations obtained from a spleen, bone marrow, lymph node or otherlymph organs, e.g., through incubating the cells with labeledanti-rabbit IgG and sorting the labeled cells using a FACSVantage SEcell sorter (Becton-Dickinson, San Jose, Calif.). In some embodiments,single or nearly single antibody-producing cells are deposited inmicrotiter plates. If the FACS system is employed, sorted cells may bedeposited after enrichment directly into a microtiter plate.

In some cases, the first antibody may have been tested in a bioassay toshow that it has a biological activity. The bioassay may determinewhether the antibody has a biological effect, e.g., an ability toinhibit an interaction between a receptor and an a ligand by eitherbinding to the receptor and blocking binding of the ligand, or bybinding to the ligand and neutralizing it, or by promoting or inhibitinga cellular phenotype, examples of which are described above. Suchbioassays are well known in the art. Bioassays useful in this method arenumerous, and include but are not limited to cellular assays in which acellular phenotype is measured, e.g., gene expression assays; and invivo assays that involve a particular animal (which, in certainembodiments may be an animal model for a condition related to thetarget).

The antibody repertoire of the animal may be sequenced by any suitablemethod. See, e.g., Reddy et al (Nat. Biotechnol. 2010 28:965-9), Fischer(MAbs. 2011 3:17-20) and Benichou et al (Immunology. 2012 135:183-91).Sequences encoding heavy and light chains may be amplified from the cDNAusing techniques well known in the art, such as Polymerase ChainReaction (PCR). See Mullis, U.S. Pat. No. 4,683,195; Mullis et al., U.S.Pat. No. 4,683,195; Polymerase Chain Reaction: Current Communication inMolecular Biology, Cold Springs Harbor Press, Cold Spring Harbor, N.Y.,1989. Briefly, cDNA segments encoding the variable domain of theantibody are exponentially amplified by performing sequential reactionswith a DNA polymerase. The reaction is primed by a 5′ primer and a 3′DNA primer. In some embodiments, the 3′ antisense primer correspondingto a DNA sequence in the constant (or joining) region of theimmunoglobulin chain and the 5′ primer (or panel of related primers)corresponding to a DNA sequence in the variable region of theimmunoglobulin chain or within the conserved leader sequence. Thiscombination of oligonucleotide primers has been used in the PCRamplification of murine immunoglobulin cDNAs of unknown sequence (seeSastry et al., Proc Natl. Acad. Sci. 86:5728-5732, 1989 and Orlandi etal., Proc. Natl. Acad. Sci. 86:3833-3837, 1989). Alternatively, an“anchored polymerase chain reaction” may be performed (see Loh et al.,Science 243:217-220, 1989). In this procedure, the first strand cDNA isprimed with a 3′ DNA primer as above, and a poly(dG tail) is then addedto the 3′ end of the strand with terminal deoxynucleotidyl transferase.The product is then amplified by PCR using the specific 3′ DNA primerand another oligonucleotide consisting of a poly(dC) tail attached to asequence with convenient restriction sites. In many embodiments,however, the entire polynucleotide encoding a heavy or light chainvariable domain is amplified using primers spanning the first and lastcodons of those regions. In certain cases, universal primers may beused. Suitable restriction sites and other tails may be engineered intothe amplification oligonucleotides to facilitate cloning and furtherprocessing of the amplification products. Amplification procedures usingnested primers may also be used, where such nested primers are wellknown to one of skill in the art. Exemplary methods for amplifyingantibody-encoding nucleic acid is also described in Wrammert (Nature2008 453: 667-672) and Scheid (Nature 2009 458: 636-640), for example.Several strategies for cloning antibody sequences by PCR are known andmay be readily adapted for use in the instant method (e.g., by using aCDR-specific primer in addition to a disclosed primer). Such strategiesinclude those described by: LeBoeuf (Gene. 1989 82:371-7), Dattamajumdar(Immunogenetics. 1996 43:141-51), Kettleborough (Eur. J. Immunol. 199323:206-11), Babcook (A novel strategy for generating monoclonalantibodies from single, isolated lymphocytes producing antibodies ofdefined specificities Proc. Natl. Acad. Sci. 1996 93: 7843-7848) andWilliams (Cold Spring Harb. Symp. Quant. Biol. 1989 54:637-47) as wellas many others. In certain cases, the second primer may be a mixture ofdifferent primers or degenerate primers, for example.

In this embodiment, the antibody-producing cells may be combined beforesequencing (in which case the initial amplification product will containa mixture of a plurality of different products that can be discriminatedby cloning the products or using single molecule sequencingtechnologies), or the cells may be kept separate from one another (inwhich case the initial amplification product amplified from a singlecell may contain a single species that can be sequenced). In particularembodiments, particularly if the antigen elicits a strong response inthe animal, the antibody repertoire may be sequenced in the absence ofany antigen-based enrichment of antibody producing cells. In theseembodiments, the method may involve: a) obtaining the antibody heavychain sequences and the antibody light chain sequences from a populationof B cells of an animal, wherein the population of B cells is notenriched for B cells that produce antibodies that specifically bind to atarget antigen. In particular embodiments, this part of the methodinvolves amplifying a population of nucleic acids that encode heavy andlight chains by PCR from cDNA made from antibody-producing cells of theanimal, and then sequencing the population of nucleic acids.

This step may comprise obtaining nucleotide sequences encoding at least1% (e.g., at least 2%, at least 5% or at least 10%) of theantigen-reactive antibody repertoire of the animal. This may beaccomplished by sequencing heavy and light chain cDNAs from 5,000, atleast 10,000, at least 50,000, or at least 100,000, or more antibodyproducing cells.

In some embodiments, the reference heavy and light chain sequences maybe deposited into a database without analysis. In other embodiments, thesequences may be analyzed, e.g., to determine whether they are heavychain sequences or light chain sequences, etc.

In the next step, the sequence obtained from the first antibody is usedto screen the reference sequences to identify antibodies that arerelated by lineage to the first antibody. The variable regions ofantibodies within a related group of antibodies have amino acidsequences that are very similar. For example, the VH or VL domains ofantibodies within a related group of antibodies may have amino acidsequences that are at least about 80% identical (e.g., at least 85%identical, at least 90% identical, at least 95% identical, at least 98%identical or at least 99% identical), ignoring any gaps or insertionsmade to facilitate alignment of the sequences. Antibodies within arelated group of antibodies have VL domains that are similar to eachother, as well as VH domains that are similar to each other. In otherwords, in certain embodiments the VH or VL domains of two differentantibodies that are related to each other by lineage usually contain upto about ten (i.e., one, two, three, four or five or more) amino aciddifferences. An amino acid difference may be present at any position ofthe variable domain, including in any CDR or in any framework region. Inthese embodiments, any suitable sequence comparison program, e.g., BLAST(Altschul et al., J. Mol. Biol. 215:403-10, 1990) comparisons may beperformed using default parameters, including choosing the BLOSUM62matrix, an expect threshold of 10, low complexity filter off, gapsallowed, and a word size of 3. A sequence comparison may be done usingDNA sequence or translated protein sequence. In some embodiments, thereference sequences may have been analyzed and placed into lineagegroups (one set of groups for the heavy chains and another set of groupsfor the light chains, if the pairing is unknown) before the sequencesare screened to identify heavy and light chains that are in the samelineage group as the heavy and light chains of the first antibody.

In some embodiments, the amino acid positions of the first antibody maybe numbered using a suitable numbering system, such as that provided byChothia (J Mol Biol 1998; 278: 457-79) or Kabat (1991, Sequences ofProteins of Immunological Interest, DHHS, Washington, D.C.). CDR and/orframework residues may be identified using these methods. Antibodiesthat are related to another by lineage also have H3 CDRs that are almostidentical, as well as L3 CDRs that are almost identical. In theseembodiments, any two antibodies that are related will have L3 and H3CDRs that are each identical in length and have near identical sequences(i.e., that contain 0, 1, 2, 3, 4 or 5 amino acid changes). In otherwords the L3 CDRs of the two antibodies are identical in length and nearidentical in sequence and the H3 CDRs of the two antibodies areidentical in length and near identical in sequence. In theseembodiments, the reference sequences may be searched for a motif, e.g.,the heavy and light chain CDR3 regions, using a motif searchingalgorithm.

Methods for identifying antibodies that are related by lineage are knownand are described in a number of publications including Magori-Cohen(Bioinformatics 2006 22: e332-40), Manske (Clin. Immunol. 2006120:106-20), Kleinstein (J. Immunol. 2003 171: 4639-49), Clement (Mol.Ecol. 2000 9: 1657-1659), Mehr (J. Immunol. 2004 172 4790-6), Wrammert(Nature 2008 453:667-672), Scheid (Nature 2009 458: 636-640), which areincorporated by reference herein for disclosure of those methods. Thefirst antibody sequence and the reference antibody sequences should allbe from a single animal, i.e., an individual mouse or an individualrabbit. In certain cases, the sequences may be aligned by eye, or byemploying an alignment program such as one of the CLUSTAL suite ofprograms (Thompson et al Nucleic Acids Research, 22:4673-4680).

As would be apparent, the sequencing may be done using a next generationsequencing platform, e.g., Illumina's reversible terminator method,Roche's pyrosequencing method (454), Life Technologies' sequencing byligation (the SOLiD platform) or Life Technologies' Ion Torrentplatform, etc. Examples of such methods are described in the followingreferences: Margulies et al (Nature 2005 437: 376-80); Ronaghi et al(Analytical Biochemistry 1996 242: 84-9); Shendure (Science 2005 309:1728); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox et al(Methods Mol Biol. 2009; 553:79-108); Appleby et al (Methods Mol Biol.2009; 513:19-39) and Morozova (Genomics. 2008 92:255-64), which areincorporated by reference for the general descriptions of the methodsand the particular steps of the methods, including all startingproducts, reagents, and final products for each of the steps. In otherembodiments, the sequencing may be done using nanopore sequencing (e.g.as described in Soni et al Clin Chem 53: 1996-2001 2007, or as describedby Oxford Nanopore Technologies).

Depending how many sequences are obtained, this analysis may result in alist of at least 5 heavy chain sequences (e.g., at least 10, at least50, at least 100, at least 1000 or at least 5,000 or more heavy chainsequences) that are related by lineage to the heavy chain of the firstantibody, and a list of at least 5 light chain sequences (e.g., at least10, at least 50, at least 100, at least 1000 or at least 5,000 or morelight chain sequences) that are related by lineage to the light chain ofthe first antibody.

After the related heavy and light chain sequences have been identified,the method may comprise testing at least one of the identified heavy andlight chain sequences to identify a second antibody that binds to thesame antigen as the first antibody. This step may involve pairing anidentified heavy chain with an identified light chain, pairing the heavychain of the first antibody with an identified heavy chain, or pairingthe light chain of the first antibody with an identified light chain. Insome cases, the heavy and light chains may be combined with each other,e.g., systematically or at random, to provide antibodies that are notproduced by the immunized animal, i.e., to provide a library ofantibodies that contains antibodies that are neither the “first”antibody or an antibody related to the first antibody by lineage.Because the first antibody and related antibodies are related by lineageand contain minimal sequence differences relative to one another, theresultant antibodies—which contain new combinations of heavy and lightchains relative to the parent and related antibodies—would be expectedto be functional (i.e., would be expected to bind the same antigen asthe first antibody). The new antibodies can be screened using standardmethods, some of which are described below, to identify an antibody witha desired activity. This antibody may contain a heavy chain from thefirst antibody and a light chain from a second antibody, where the firstand second antibodies are different antibodies that are related bylineage. In particular embodiments, this step may comprise testing atleast 10% (e.g., at least 20%, at least 30% or at least 50%) of allpossible combinations of identified heavy and light chains for bindingto the antigen.

Depending on how the sequencing is done, the sequences encoding theantibodies may be made synthetically, or they may be amplified by PCRfrom a cDNA sample made from the animal. In either case, sequencesencoding heavy and light chains may cloned into suitable vectors andexpressed in a suitable host cells. Vector systems and host cells forproducing antibodies are well known.

As would be readily apparent, the pairing of the heavy and light chainsmay be done many different ways, e.g., systematically or randomly and,in certain cases, may be done using pooled nucleic acid. In particularembodiments, the pairing may involve systematically combining thevariable domains of the heavy and light chains of the first antibody andthe further antibodies to produce a library of antibodies that containsat least 50% of all possible combinations of variable domains. In otherembodiments, the pairing step may involve: i. introducing: a) a pool ofheavy chain-encoding nucleic acid that encodes a plurality of differentamplified heavy chain variable domains and b) a pool of lightchain-encoding nucleic acid that encodes a plurality of differentamplified light chain variable domains, into population of cells, andii. selecting cells that contain both a heavy chain-encoding nucleicacid and a light chain-encoding nucleic acid, to produce a library ofcells that produce a library of antibodies. As would be apparent, anumber of different cloning strategies may be employed to produce poolsof nucleic acids.

In some embodiments, the heavy and light chains can be paired and testedin a combinatorial manner using phage display methods (see, generally,Smith et al Chem. Rev. 1997 97: 391-410). In these embodiments, theheavy and light chains may be cloned into a phage display vector suchthat the resultant phage display library contains at least 90% of thepossible combinations of heavy and light chains. The new antibodies canthen be produced and tested using phage display methods, methods forwhich are known.

In particular embodiments, the second antibody may contain naturallypaired heavy and light chain variable domains, or non-naturally pairedheavy and light chain variable domains (i.e., heavy and light chainvariable domains from different antibodies of the same lineage group).Since the antibodies are from the same lineage group, it is expectedthat such antibodies will be functional. In particular embodiments, thepairing of the heavy and light chains may be systematic (e.g., everyheavy chain is tested in combination with every light chain) or random(e.g., every heavy chain is tested with randomly selected light chains),for example.

In some embodiments, the method may comprise testing the second antibodyto determine the specificity or affinity to the antigen. A secondantibody may inhibit at least one activity of its target in the range ofabout 20% to 100%, e.g., by at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, usually up toabout 70%, up to about 80%, up to about 90% or more. In certain assays,a subject antibody may inhibit its target with an IC₅₀ of 1×10⁻⁷M orless (e.g., 1×10⁻⁷M or less, 1×10⁻⁸M or less, 1×10⁻⁹M or less, usuallyto 1×10⁻¹² M or 1×10⁻¹³M). In assays in which a mouse is employed, asubject antibody may have an ED₅₀ of less than 1 μg/mouse (e.g., 10ng/mouse to about 1 μg/mouse). In certain embodiments, a subjectantibody may be contacted with a cell in the presence of a ligand, and aligand response phenotype of the cell is monitored. The method maycomprise humanizing the second antibody, methods for performing whichare known.

Methods for Producing Antibodies

In many embodiments, the nucleic acids encoding a subject monoclonalantibody are introduced directly into a host cell, and the cellincubated under conditions sufficient to induce expression of theencoded antibody.

Any cell suitable for expression of expression cassettes may be used asa host cell. For example, yeast, insect, plant, etc., cells. In manyembodiments, a mammalian host cell line that does not ordinarily produceantibodies is used, examples of which are as follows: monkey kidneycells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7,ATCC CRL 165 1); human embryonic kidney cells (HEK-293, Graham et al. J.Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. (USA) 77:4216, (1980); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70);african green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al.,Annals N. Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658);and mouse L cells (ATCC CCL-1). Additional cell lines will becomeapparent to those of ordinary skill in the art. A wide variety of celllines are available from the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209.

Methods of introducing nucleic acids into cells are well known in theart. Suitable methods include electroporation, particle gun technology,calcium phosphate precipitation, direct microinjection, and the like.The choice of method is generally dependent on the type of cell beingtransformed and the circumstances under which the transformation istaking place (i.e. in vitro, ex vivo, or in vivo). A general discussionof these methods can be found in Ausubel, et al, Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995. In some embodimentslipofectamine and calcium mediated gene transfer technologies are used.

After the subject nucleic acids have been introduced into a cell, thecell is typically incubated, normally at 37° C., sometimes underselection, for a period of about 1-24 hours in order to allow for theexpression of the antibody. In most embodiments, the antibody istypically secreted into the supernatant of the media in which the cellis growing in.

In mammalian host cells, a number of viral-based expression systems maybe utilized to express a subject antibody. In cases where an adenovirusis used as an expression vector, the antibody coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a nonessential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the antibody molecule ininfected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA81:355-359 (1984)). The efficiency of expression may be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544(1987)).

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule may be engineered.

Rather than using expression vectors which contain viral origins ofreplication, host cells can be transformed with immunoglobulinexpression cassettes and a selectable marker. Following the introductionof the foreign DNA, engineered cells may be allowed to grow for 1-2 daysin an enriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into achromosome and grow to form foci which in turn can be cloned andexpanded into cell lines. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that interact directlyor indirectly with the antibody molecule.

Once an antibody molecule of the invention has been produced, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In many embodiments, antibodies are secretedfrom the cell into culture medium and harvested from the culture medium.

Determining Binding Affinity of an Antibody

Once a modified antibody is produced, it may be tested for affinityusing any known method, such as: 1) competitive binding analysis using alabeled (radiolabeled or fluorescent labeled) parent antibody, amodified antibody and an antigen recognized by the parent antibody; 2)surface plasmon resonance using e.g. BIACore instrumentation to providethe binding characteristics of an antibody. Using this method antigensare immobilized on solid phase chips and the binding of antibodies inliquid phase are measured in a real-time manner; 3) flow cytometry, forexample, by using fluorescent activated cell sorting (FACS) analysis tostudy antibody binding to cell surface antigens; 4) ELISA; or 5)equilibrium dialysis. Methods for measuring binding affinity aregenerally described in Harlow et al., Antibodies: A Laboratory Manual,First Edition (1988) Cold Spring Harbor, N.Y.; Ausubel, et al, ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995).

If affinity analysis reveals a decrease in antibody binding for themodified antibody as compared to its parent antibody, “fine tuning” maybe performed to increase the affinity. One method of doing this is tosystematically change back each modified residue by site-directedmutagenesis. By expressing and analyzing these back mutant antibodies,one would predict the key residues that cannot be modified withoutdecreasing affinity.

Utility

An antibody produced by the present method finds use in diagnostics, inantibody imaging, and in treating diseases treatable by monoclonalantibody-based therapy. In particular, an antibody produced by themethod described above may be humanized and used for passiveimmunization or the removal of unwanted cells or antigens, such as bycomplement mediated lysis or antibody mediated cytotoxicity (ADCC), allwithout substantial immune reactions (e.g., anaphylactic shock)associated with many prior antibodies. For example, the antibodies ofthe present invention may be used as a treatment for a disease where thesurface of an unwanted cell specifically expresses a protein recognizedby the antibody (e.g. HER2, or any other cancer specific marker) or theantibodies may be used to neutralize an undesirable toxin, irritant orpathogen. Humanized antibodies are particularly useful for the treatmentof many types of cancer, for example colon cancer, lung cancer, breastcancer prostate cancer, etc., where the cancers are associated withexpression of a particular cellular marker. Since most, if not all,disease-related cells and pathogens have molecular markers that arepotential targets for antibodies, many diseases are potentialindications for humanized antibodies. These include autoimmune diseaseswhere a particular type of immune cells attack self-antigens, such asinsulin-dependent diabetes mellitus, systemic lupus erythematosus,pernicious anemia, allergy and rheumatoid arthritis; transplantationrelated immune activation, such as graft rejection and graft-vs-hostdisease; other immune system diseases such as septic shock; infectiousdiseases, such as viral infection or bacteria infection; cardiovasculardiseases such as thrombosis and neurological diseases such asAlzheimer's disease. An antibody of particular interest is one thatmodulates, i.e., reduces or increases a symptom of diseases such asseptic shock; infectious diseases, such as viral infection or bacteriainfection; cardiovascular diseases such as thrombosis and neurologicaldiseases such as Alzheimer's disease.

An antibody of particular interest is one that modulates, i.e., reducesor increases a symptom of the animal model disease or condition by atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 90%, ormore, when compared to a control in the absence of the antibody. Ingeneral, a monoclonal antibody of interest will cause a subject animalto be more similar to an equivalent animal that is not suffering fromthe disease or condition. Monoclonal antibodies that have therapeuticvalue that have been identified using the methods and compositions ofthe invention are termed “therapeutic” antibodies.

Kits

Also provided by the subject invention are kits for practicing thesubject methods, as described above. The subject kits at least includeone or more of: an antibody made according to the above methods, anucleic acid encoding the same, or a cell containing the same. Theantibody may be humanized. Other optional components of the kit include:restriction enzymes, control primers and plasmids; buffers; etc. Thenucleic acids of the kit may also have restrictions sites, multiplecloning sites, primer sites, etc to facilitate their ligation tonon-rabbit antibody CDR encoding nucleic acids. The various componentsof the kit may be present in separate containers or certain compatiblecomponents may be precombined into a single container, as desired. Inaddition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Also provided by the subject invention is are kits including at least acomputer readable medium including programming as discussed above andinstructions. The instructions may include installation or setupdirections. The instructions may include directions for use of theinvention with options or combinations of options as described above. Incertain embodiments, the instructions include both types of information.

Providing the software and instructions as a kit may serve a number ofpurposes. The combination may be packaged and purchased as a means forproducing rabbit antibodies that are less immunogenic in a non-rabbithost than a parent antibody, or nucleotide sequences them.

The instructions are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc, including the samemedium on which the program is presented.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

That which is claimed is:
 1. A method of screening, comprising: a)obtaining the nucleotide sequences of: i. a VH cDNA, wherein said VHcDNA encodes the variable domain of a heavy chain of a first antibody ofan animal; and ii. a VL cDNA, wherein said VL cDNA encodes the variabledomain of a light chain of said first antibody; b) obtaining at least amillion nucleotide sequences of cDNAs encoding at least a portion of theantibody repertoire of said animal; c) computationally screening thesequences obtained in b) to identify heavy and light chain sequencesthat are related by lineage to the heavy and light chain sequences ofa); and d) testing in vitro at least one pair of the heavy and lightchain sequences identified in c) to identify a second antibody thatbinds to the same antigen as the first antibody.
 2. The method of claim1, wherein the testing step d) comprises testing at least 10% of allpossible combinations of heavy and light chains identified in step c)for binding to said antigen.
 3. The method of claim 1, wherein the heavyand light chain sequences tested in step d) are randomly selected fromthe heavy and light chain sequences identified in step b).
 4. A methodof producing an antibody, the method comprising a) obtaining thenucleotide sequences of: i. a heavy chain-encoding nucleic acid thatencodes the variable domain of a heavy chain of a first antibody of ananimal; and ii. a light chain-encoding nucleic acid that encodes thevariable domain of a light chain of the first antibody; b) obtaining atleast a million nucleotide sequences of cDNAs encoding at least aportion of the antibody repertoire of the animal; c) computationallyscreening the sequences obtained in b) to identify heavy and light chainsequences that are related by lineage to the heavy and light chainsequences of a); d) introducing at least one pair of the heavy and lightchain sequences obtained in c) into a host cell in vitro; e) incubatingthe host cell to permit expression of the antibody; f) purifying theantibody expressed in e) to produce a second antibody that binds to thesame antigen as the first antibody.
 5. The method according to claim 1,wherein the obtaining step b) comprises sequencing cDNAs encoding heavyand light chains made from splenocytes of said animal or fromcirculating B cells of said animal.
 6. The method according to claim 1,wherein the obtaining step b) comprises amplifying a population ofnucleic acids that encode heavy and light chains by PCR from cDNA madefrom antibody producing cells of said animal, and then sequencing saidpopulation of nucleic acids.
 7. The method of claim 6, wherein saidantibody-producing cells are not pre-selected by their ability toproduce an antibody to said antigen.
 8. The method according to claim 1,wherein said animal has been immunized with said antigen, multipletimes, in the presence of an adjuvant.
 9. The method according to claim1, wherein said animal has an autoimmune disease, or has resistance toor has recovered from a disease.
 10. The method of claim 1, wherein stepb) comprises obtaining nucleotide sequences encoding at least 10% of theantibody repertoire of said animal.
 11. The method of claim 1, whereinsaid computationally screening step c) is by comparing a sequence of atleast 50 contiguous amino acids of said heavy and light chains with thesequences obtained in b).
 12. The method of according to claim 1,wherein said computationally screening step c) comprises scanning thesequences obtained in step b) to identify those that encode the heavyand light chain CDR3 sequences of said first antibody.
 13. The method ofclaim 1, comprising testing said second antibody to determine thespecificity or affinity to said antigen.
 14. The method of claim 1,wherein said animal is a rabbit, human, mouse or chicken.
 15. The methodof claim 1, further comprising humanizing said second antibody.